U.S. patent number 8,394,914 [Application Number 12/460,655] was granted by the patent office on 2013-03-12 for functional polyglycolide nanoparticles derived from unimolecular micelles.
This patent grant is currently assigned to Board of Trustees of Michigan State University. The grantee listed for this patent is Gregory L. Baker, Milton R. Smith, III, Erin Vogel. Invention is credited to Gregory L. Baker, Milton R. Smith, III, Erin Vogel.
United States Patent |
8,394,914 |
Baker , et al. |
March 12, 2013 |
Functional polyglycolide nanoparticles derived from unimolecular
micelles
Abstract
Poly(glycolide) polymers are disclosed. The polymers generally
include a glycolide-based polymer backbone that includes one or
more functional groups such as alkynyl groups, hydrophilic organic
triazole groups, hydrophobic organic triazole groups (also
including amphiphilic organic triazole groups), di-triazole organic
crosslinking groups, and triazole-substituted drug derivatives. The
alkynyl groups provide reactive sites for further functionalization
of the polymer, for example by reaction with azide derivatives. The
polymers can further encapsulate a drug for delivery to a patient
(i.e., as compared to drug derivatives that are covalently attached
to the polymer). The polymers can be in the form of
thermodynamically stable unimolecular micelles or crosslinked
nanoparticles. The polymer compositions are completely
biodegradable and hold great potential for use in biomedical
applications.
Inventors: |
Baker; Gregory L. (Haslett,
MI), Smith, III; Milton R. (East Lansing, MI), Vogel;
Erin (Midland, MI) |
Applicant: |
Name |
City |
State |
Country |
Type |
Baker; Gregory L.
Smith, III; Milton R.
Vogel; Erin |
Haslett
East Lansing
Midland |
MI
MI
MI |
US
US
US |
|
|
Assignee: |
Board of Trustees of Michigan State
University (East Lansing, MI)
|
Family
ID: |
41447934 |
Appl.
No.: |
12/460,655 |
Filed: |
July 22, 2009 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090325292 A1 |
Dec 31, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12229544 |
Aug 25, 2008 |
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61135679 |
Jul 23, 2008 |
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60966042 |
Aug 24, 2007 |
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Current U.S.
Class: |
528/354 |
Current CPC
Class: |
A61K
47/6907 (20170801); A61K 9/1075 (20130101); C08G
63/08 (20130101); A61K 47/34 (20130101); A61K
47/6935 (20170801); A61K 9/5153 (20130101); A61K
47/6931 (20170801); C08G 63/6852 (20130101); B82Y
5/00 (20130101) |
Current International
Class: |
C08G
63/08 (20060101) |
Field of
Search: |
;435/375 ;424/400
;528/354,271 |
References Cited
[Referenced By]
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|
Primary Examiner: Listvoyb; Gregory
Attorney, Agent or Firm: Harness, Dickey & Pierce,
P.L.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
Priority to U.S. Provisional Application Ser. No. 61/135,679, filed
Jul. 23, 2008, which is incorporated herein by reference in its
entirety, is claimed.
This application is a continuation-in-part of U.S. patent
application Ser. No. 12/229,544, filed Aug. 25, 2008 (Monday),
which in turn claims the priority benefit of U.S. Provisional
Application No. 60/966,042, filed Aug. 24, 2007, both of which are
incorporated herein by reference in their entireties.
Claims
What is claimed is:
1. A poly(glycolide) polymer comprising: a triazole reaction
product of (a) an alkynyl glycolide according to Formula III
##STR00007## (b) an azide-substituted hydrophilic organic compound;
and, (c) optionally one or more of an azido organic crosslinker, an
azide-substituted hydrophobic organic compound, an
azide-substituted liquid crystal mesogen, an azide-substituted dye,
and an azide-substituted drug; wherein: (i) R.sub.1 comprises one
or more moieties selected from a hydrogen, an alkyl group, and an
alkynyl group; (ii) R.sub.2 comprises an alkynyl group; (iii) R
comprises a terminal group; (iv) x ranges from 0 to less than 1;
and, (v) n ranges from about 10 to about 1000.
2. The poly(glycolide) polymer of claim 1, wherein the alkynyl
group comprises 3 to 12 carbon atoms.
3. The poly(glycolide) polymer of claim 1, wherein the
azide-substituted hydrophilic organic compound comprises a compound
selected from the group consisting of an azide-substituted
polyoxyalkylene, an azide-substituted organic amine, an
azide-substituted carboxylic acid, an azide-substituted carboxylate
salt, an azide-substituted alkyl polyoxyalkylene, an
azide-substituted ketone, an azide-substituted alcohol, an
azide-substituted ester, and combinations thereof.
4. The poly(glycolide) polymer of claim 1, wherein the polymer is
in the form of a unimolecular micelle having (i) a diameter of
about 50 nm or less as measured by dynamic light scattering in
aqueous solution with (ii) a substantially hydrophilic exterior and
a substantially hydrophobic interior.
5. The poly(glycolide) polymer of claim 4, wherein the unimolecular
micelle further-comprises an encapsulated drug within the
interior.
6. The poly(glycolide) polymer of claim 4, wherein the triazole
reaction product comprises moieties resulting from the reaction of
the alkynyl glycolide according to Formula III and the
azide-substituted drug.
7. The poly(glycolide) polymer of claim 1, wherein: the triazole
reaction product comprises moieties resulting from the reaction of
the alkynyl glycolide according to Formula III and the azido
organic crosslinker, the polymer is in the form of an
intramolecularly crosslinked nanoparticle having (i) a diameter of
about 35 nm or less as measured by dynamic light scattering in
aqueous solution with (ii) a substantially hydrophilic exterior and
a substantially hydrophobic interior.
8. The poly(glycolide) polymer of claim 7, wherein the crosslinked
nanoparticle further comprises an encapsulated drug within the
interior.
9. The poly(glycolide) polymer of claim 7, wherein the triazole
reaction product comprises moieties resulting from the reaction of
the alkynyl glycolide according to Formula III and the
azide-substituted drug.
10. A plurality of crosslinked nanoparticles according to claim 7,
wherein the plurality of crosslinked nanoparticles has a
distribution of crosslinking degrees selected to control the rate
of release of an encapsulated component or a covalently bound
component in the plurality of crosslinked nanoparticles.
Description
BACKGROUND OF THE DISCLOSURE
1. Field of the Disclosure
Degradable comb polymers that individually self-aggregate into
unimolecular micelles in an aqueous solution are disclosed. The
compositions generally include poly(glycolide) polymers having
pendant alkynyl groups that can be functionalized via triazole
formation with hydrophilic and other groups. The polymeric micelles
can be chemically cross-linked to form organic nanoparticles that
retain their chemical functionality and their degradability. The
micelles are capable of hydrophobic drug encapsulation and have
pendant reactive sites that can be used for cross-linking or
covalent drug attachment. The micelles are completely biodegradable
and can be used in biomedical applications for controlled drug
release.
2. Brief Description of Related Technology
Inorganic nanoparticles have a long and rich history, having
benefited from favorable physical properties such as mechanical,
thermal, and chemical stability. While comparable developments in
organic nanoparticles are more recent, the structural diversity of
organic materials and their application to complex problems in
medicine has made the synthesis and characterization of organic
nanoparticles one of the most active topics in polymer
science..sup.1
Organic nanoparticles are important in many applications, but
especially for the encapsulation of small molecules for the
delivery of therapeutic agents, personal care products, and
colorants..sup.2 Until recently, nearly all applications were based
on nanoparticles prepared by spray drying or an oil in water
emulsion approach, using homopolymers as the host. The need for
"smart" particles in medical applications, to control release
rates, target specific sites in the body, and to transport large
biomolecules such as single strand RNA for gene therapy, instigated
interest in more complicated polymer architectures and
function..sup.3-5 A variety of architectural motifs were
synthesized including many varieties of linear homopolymers and
block copolymers, and branched polymers such as stars, combs,
dendrimers, and hyper-branched structures that provide multiple
sites (usually at chain ends) for tethering molecules. In contrast
to traditional micelles, these structures are "unimolecular", and
correspond to a single molecular entity formed from individual
polymer chains and are unaffected by solution
concentration..sup.6
FIGS. 1A and 1B contrast unimolecular micelles (FIG. 1B) to
traditional micelles (FIG. 1A) derived from amphiphilic block
copolymers. In aqueous solutions, both assemble to form a
hydrophilic (blue) exterior 2 and a hydrophobic (red) interior 1
which allows encapsulation of hydrophobic materials. Traditional
polymer micelles are derived from low-cost block copolymers, but
their aggregation numbers vary and block copolymers often lack
functional groups for further elaboration. In addition, the
critical micelle concentration (cmc) defines their lower
concentration limit for in vivo applications. At low
concentrations, as in the blood stream, the equilibrium between the
micellar structure and individual surfactant molecules complicates
controlled drug release and causes potential toxicity
problems..sup.7 These limitations have been overcome by
cross-linked micelles to form stable core shell
structures,.sup.8-17 and introducing chemical functionality to the
individual molecules that of the micelle to allow introduction of
moieties for cell recognition and imaging. Attaching individual
hydrophobic and hydrophilic segments to a polymer backbone creates
a brush-like copolymer capable of forming "unimolecular micelles."
Comb polymers seem particularly promising since they can be
prepared by controlled polymerization methods and the functional
group density can be very high (e.g., about 1 functional
group/monomer repeat unit).
There have been several recent examples of cross-linked
nanoparticles synthesized by cross-linking well-defined single
polymer chains. Using a dilute solution approach Hawker.sup.18 and
Harth.sup.19 cross-linked linear styrene-vinylbenzocyclobutene
copolymers and obtained .about.6 nm nanoparticles. Harth extended
the o-quinodimethane chemistry.sup.19 by--copolymerizing styrene
and 5-vinyl-1,3-dihydrobenzo[c]thiophene, extruding SO.sub.2 to
form the highly reactive o-quinodimethane intermediate and
crosslink the polymer.
Thus, there exists a need for an improved drug delivery composition
that can serve as a vehicle for delivering a drug in a variety of
formats (e.g., encapsulated, covalently attached) in a controlled
release manner.
SUMMARY
As part of ongoing interest in designing, synthesizing, and
characterizing functional polylactides,.sup.20-25 the synthesis and
characterization of poly(propargyl glycolide) (poly(PGL) was
recently reported..sup.26 Poly(PGL) is a comb polymer precursor
that allows facile elaboration of the polylactide backbone via
"click" chemistry: the Cu(I)-mediated 1,3-dipolar cycloaddition of
azides to alkynes. Poly(PGL) "base" polymers were synthesized with
molecular weights predicted by the monomer to initiator ratio, and
polydispersities that ranged from 1.1-1.5. After full
characterization of the base polymer, alkyl and PEO-based azides
were covalently attached to the polymer backbone to obtain
amphiphilic comb polymers that exhibit lower critical solution
transitions over a broad temperature range. The mild conditions
developed for the "click" chemistry, generating Cu(I) in situ via
copper sulfate pentahydrate and sodium ascorbate in DMF at room
temperature, precluded degradation of the sensitive polylactide
backbone. This subject matter is disclosed in the related Baker et
al. U.S. Provisional Application No. 60/966,042, which is
incorporated herein by reference in its entirety.
Poly(glycolide) polymers are disclosed. The polymers generally
include a glycolide-based polymer backbone that includes one or
more functional groups such as alkynyl groups, hydrophilic organic
triazole groups, hydrophobic organic triazole groups, di-triazole
organic crosslinking groups, and triazole-substituted drug
derivatives. The alkynyl groups provide reactive sites for further
functionalization of the polymer, for example by reaction with
azide derivatives. Alkynyl and azide groups react via the "click"
chemistry mechanism to form functional groups covalently bonded to
the polymer via a triazole link, for example hydrophilic organic
groups, hydrophobic organic groups, crosslinking groups, and/or
drug derivatives.
The polymers can be in the form of unimolecular micelles or
crosslinked nanoparticles. The polymers can further encapsulate a
drug for delivery to a patient (i.e., as compared to drug
derivatives that are covalently attached to the polymer).
Thermodynamically stable unimolecular micelles are readily
prepared, and can be further crosslinked to form unimolecular
crosslinked nanoparticles. The resulting compositions are
completely biodegradable in both the unimolecular micelle and
crosslinked nanoparticle form. The flexibility of click
functionalization and crosslinking allows the design of
compositions applicable to a wide variety of controlled-release
drug delivery applications.
In a first embodiment, a poly(glycolide) polymer comprises: one or
more repeating units according to Formula I
##STR00001## wherein: (i) R.sub.1 comprises one or more moieties
selected from an alkynyl group, a hydrophilic organic triazole
group, a hydrophobic organic triazole group, a triazole organic
crosslinking group (e.g., di-triazole organic crosslinking group),
a triazole-substituted liquid crystal mesogen, a
triazole-substituted dye, and a triazole-substituted drug
derivative; (ii) R.sub.2 comprises one or more moieties selected
from a hydrogen, an alkyl group, an alkynyl group, a hydrophilic
organic triazole group, a hydrophobic organic triazole group, a
triazole organic crosslinking group (e.g., di-triazole organic
crosslinking group), a triazole-substituted liquid crystal mesogen,
a triazole-substituted dye, and a triazole-substituted drug
derivative; and, (iii) x is between 0 and 1.
In variations of the first embodiment, the alkynyl group comprises
3 to 12 carbon atoms (e.g., a propargyl group having a terminal
alkynyl group), the alkyl group comprises 1 to 18 carbon atoms, the
hydrophilic organic triazole group comprises a triazole reaction
product of an alkynyl group and an azide-substituted
polyoxyethylene (e.g., the triazole reaction product of a propargyl
group and azidoethyl tetraethylene glycol methyl ether), the
hydrophobic organic triazole group comprises a triazole reaction
product of an alkynyl group and an azide-substituted alkane (e.g.,
the triazole reaction product of a propargyl group and
1-azidodecane), the triazole organic crosslinking group comprises a
di-triazole reaction product of two alkynyl groups and a diazido
alkane (e.g., the di-triazole reaction product of two propargyl
groups and a 1,5-diazidopentane), and/or the triazole-substituted
drug derivative comprises a triazole reaction product of an alkynyl
group and an azide-substituted drug. Alternatively, the hydrophilic
organic triazole group comprises a triazole reaction product of an
alkynyl group and a moiety selected from the group consisting of an
azide-substituted polyoxyethylene polyoxyalkylene, an
azide-substituted organic amine, an azide-substituted carboxylic
acid, an azide-substituted carboxylate salt, an azide-substituted
alkyl polyoxyalkylene, an azide-substituted ketone, an
azide-substituted alcohol, an azide-substituted ester, and
combinations thereof (e.g., the hydrophilic organic triazole group
can include a distribution of multiple types of groups, with a
single group attached at each R.sub.1 or R.sub.2 location).
Preferably, R.sub.1 is the hydrophilic organic triazole group and x
ranges from 0.5 to 0.95, and R.sub.2 comprises the di-triazole
organic crosslinking group (and optionally the triazole-substituted
drug derivative in addition). The poly(glycolide) polymer can be in
the form of a copolymer (e.g., random or block) that further
comprises lactide repeating units.
In a second embodiment, a poly(glycolide) polymer comprises: one or
more polymeric chains according to Formula II
##STR00002## wherein: (i) R.sub.1 comprises one or more moieties
selected from an alkynyl group and a triazole organic crosslinking
group (e.g., di-triazole organic crosslinking group); (ii) R.sub.2
comprises a hydrophilic organic triazole group and optionally one
or more moieties selected from a hydrogen, an alkyl group, an
alkynyl group, a hydrophobic organic triazole group, a
triazole-substituted liquid crystal mesogen, a triazole-substituted
dye, and a triazole-substituted drug derivative; (iii) R comprises
a terminal group (e.g., a hydrogen or an alkyl group); (iv) x
ranges from 0 to less than 1; and, (v) n ranges from about 10 to
about 1000.
In variations of the second embodiment, the alkynyl group comprises
3 to 12 carbon atoms (e.g., a propargyl group having a terminal
alkynyl group), the alkyl group comprises 1 to 18 carbon atoms, the
hydrophilic organic triazole group comprises a triazole reaction
product of an alkynyl group and an azide-substituted
polyoxyethylene and/or any of the above azide-substituted
hydrophilic groups (e.g., the triazole reaction product of a
propargyl group and azidoethyl tetraethylene glycol methyl ether),
the hydrophobic organic triazole group comprises a triazole
reaction product of an alkynyl group and an azide-substituted
alkane (e.g., the triazole reaction product of a propargyl group
and 1-azidodecane), the triazole organic crosslinking group
comprises a di-triazole reaction product of two alkynyl groups and
a diazido alkane (e.g., the di-triazole reaction product of two
propargyl groups and a 1,5-diazidopentane), and/or the
triazole-substituted drug derivative comprises a triazole reaction
product of an alkynyl group and an azide-substituted drug. The
polymer can be in the form of a unimolecular micelle having a
diameter of about 50 nm or less (e.g., about 15 nm to about 50 nm;
as measured by dynamic light scattering in aqueous solution) with a
substantially hydrophilic exterior and a substantially hydrophobic
interior. The polymer also can be in the form of an
intramolecularly crosslinked nanoparticle having a diameter of
about 35 nm or less (e.g., about 8 nm to about 35 nm; as measured
by dynamic light scattering in aqueous solution) with a
substantially hydrophilic exterior and a substantially hydrophobic
interior when R.sub.1 comprises the di-triazole organic
crosslinking group and x is larger than 0. The polymer also can
comprise a plurality of crosslinked nanoparticles having a
distribution of crosslinking degrees selected to control the rate
of release of an encapsulated component or a covalently bound
component in the plurality of crosslinked nanoparticles. Either the
unimolecular micelle or the crosslinked nanoparticle can further
comprise an encapsulated drug within the interior, or a covalently
bound triazole-substituted drug derivative.
In a third embodiment, a poly(glycolide) polymer comprises: a
triazole reaction product of (a) an alkynyl glycolide according to
Formula III
##STR00003## (b) an azide-substituted hydrophilic organic compound;
and, (c) optionally one or more of a azido organic crosslinker
(e.g., a diazido organic crosslinker), an azide-substituted
hydrophobic organic compound, an azide-substituted liquid crystal
mesogen, an azide-substituted dye, and an azide-substituted drug;
wherein: (i) R.sub.1 comprises one or more moieties selected from a
hydrogen, an alkyl group, and an alkynyl group; (ii) R.sub.2
comprises an alkynyl group; (iii) R comprises a terminal group
(e.g., a hydrogen or an alkyl group); (iv) x ranges from 0 to less
than 1; and, (v) n ranges from about 10 to about 1000.
In variations of the third embodiment, the alkynyl group comprises
3 to 12 carbon atoms (e.g., a propargyl group having a terminal
alkynyl group), the alkyl group comprises 1 to 18 carbon atoms,
and/or the azide-substituted hydrophilic organic compound comprises
an azide-substituted polyoxyethylene (e.g., azidoethyl
tetraethylene glycol methyl ether or any of the above general
azide-substituted hydrophilic organic compounds). The polymer can
be in the form of a unimolecular micelle having a diameter of about
50 nm or less (e.g., about 15 nm to about 50 nm; as measured by
dynamic light scattering in aqueous solution) with a substantially
hydrophilic exterior and a substantially hydrophobic interior. The
polymer also can be in the form of an intramolecularly crosslinked
nanoparticle having a diameter of about 35 nm or less (e.g., about
8 nm to about 35 nm; as measured by dynamic light scattering in
aqueous solution) with a substantially hydrophilic exterior and a
substantially hydrophobic interior when the triazole reaction
product comprises moieties resulting from the reaction of the
alkynyl glycolide according to Formula III and the azido organic
crosslinker. The polymer also can comprise a plurality of
crosslinked nanoparticles having a distribution of crosslinking
degrees selected to control the rate of release of an encapsulated
component or a covalently bound component in the plurality of
crosslinked nanoparticles. Either the unimolecular micelle or the
crosslinked nanoparticle can further comprise an encapsulated drug
within the interior, or a covalently bound triazole-substituted
drug derivative.
Also disclosed is a method of delivering a drug-compound to a cell,
the method comprising: (a) providing the drug compound with the
poly(glycolide) polymer of any of the foregoing embodiments; and,
(b) releasing the drug compound to the cell over a period of time.
The cell can be in vivo or in vitro, and the poly(glycolide)
polymer can be in the form of a unimolecular micelle or an
intramolecularly crosslinked nanoparticle. In an embodiment, the
drug compound comprises a triazole-substituted drug derivative
covalently bound to the poly(glycolide) polymer. In another
embodiment, the drug compound is encapsulated by the
poly(glycolide) polymer. In yet another embodiment, the drug
compound is in dosage unit form with a carrier.
Additional features of the disclosure may become apparent to those
skilled in the art from a review of the following detailed
description, taken in conjunction with the drawings, examples, and
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
For a more complete understanding of the disclosure, reference
should be made to the following detailed description and
accompanying drawings wherein:
FIGS. 1a and 1b are schematics comparing a traditional micelle
formed from block copolymers (FIG. 1a) and the formation of a
unimolecular micelle (FIG. 1b) from an amphiphilic comb polymer in
aqueous solution. The hydrophilic (blue; 1) chains are soluble in
the aqueous phase, while hydrophobic segments (red; 2) collapse
into the interior of the micelle.
FIG. 2 illustrates the synthesis and crosslinking of unimolecular
micelles from propargyl glycolide, azidoethyl tetraethylene glycol
methyl ether, and 1,5-diazidopentane.
FIG. 3a is an AFM image of unimolecular micelles prepared by
spin-coating a 0.20 mg/ml solution onto a silicon wafer.
FIG. 3b is a TEM (transmission electron microscope) image of
unimolecular micelles deposited from a 0.20 mg/ml solution onto a
FORMVAR resin-coated Ni grid and allowed to settle for 2 min before
removing the excess solution. The sample was negatively stained
with 1% phosphotungstic acid; scale bar=100 nm.
FIG. 4a illustrates the crosslinking reaction of FIG. 2 (pendant
hydrophilic organic groups omitted for clarity).
FIG. 4b is a TEM image of crosslinked nanoparticles (click reaction
products of poly(propargyl glycolide) (72,000 g/mol) with
1,5-diazidopentane) that were deposited onto a FORMVAR resin-coated
nickel grid from a 0.25 mg/ml aqueous solution, and negatively
stained with phosphotungstic acid after solvent removal.
FIG. 4c illustrates the IR spectra of crosslinked nanoparticles as
a function of % crosslinking of free alkynyl groups after
functionalization with a polyoxyethylene azide (% crosslinking
degrees of 0% ("poly(PGL"), 46%, 73%, and 100%).
FIG. 4d is an AFM (atomic force microscope) image of crosslinked
nanoparticles prepared by spin-coating a 0.20 mg/ml solution onto a
silicon wafer.
FIG. 4e is a TEM image of crosslinked nanoparticles deposited from
a 0.20 mg/ml solution (370,000.times. magnification; scale bar=100
nm).
FIG. 5a is a schematic illustrating two different reaction paths
for the covalent binding of an azide-substituted drug to a
crosslinked nanoparticle.
FIG. 5b illustrates the fluorescence intensity of crosslinked
nanoparticles having an azide-substituted fluorescent dye precursor
bound to the crosslinked nanoparticle according to the different
reaction paths of FIG. 5a.
FIG. 6 illustrates the UV-vis spectra of: azobenzene-loaded
unimolecular polymeric micelles (blue; line 1), unimolecular
polymeric micelles (green; line 2), and azobenzene in water (red;
line 3).
FIG. 7 illustrates the controlled released of azobenzene from
azobenzene-loaded crosslinked nanoparticles (solid squares) and
azobenzene-loaded unimolecular polymeric micelles (open squares)
suspended in water at room temperature over about 10 hours.
FIG. 8a illustrates cortical astrocytes cultured in media
containing 10 .mu.g/ml of crosslinked nanoparticles for 24
hours.
FIG. 8b compares the cell toxicity (in normalized relative
fluorescence units) for various nanoparticle solutions both
according to the disclosure and using commercially available
reference nanoparticles.
FIG. 9 compares the relative rates of hydrolytic degradation of
polyoxyethylene-functionalized poly(propargyl glycolide) (open
squares) and rac-lactide (open triangles) by measuring the
molecular weight as a function of time.
FIG. 10 illustrates representative chemical structures of
azide-substituted organic compounds capable of
click-functionalization on the poly(glycolide) polymer.
FIG. 11 illustrates specific chemical structures of
azide-substituted organic compounds used to form crosslinked
poly(glycolide) polymer nanoparticles.
While the disclosed compositions and methods are susceptible of
embodiments in various forms, specific embodiments of the
disclosure are illustrated in the drawings (and will hereafter be
described) with the understanding that the disclosure is intended
to be illustrative, and is not intended to limit the claims to the
specific embodiments described and illustrated herein.
DETAILED DESCRIPTION
Poly(glycolide) polymers are disclosed. The polymers generally
include a glycolide-based polymer backbone that includes one or
more functional groups such as alkynyl groups, hydrophilic organic
triazole groups, hydrophobic organic triazole groups (also
including amphiphilic organic triazole groups), di-triazole organic
crosslinking groups, and triazole-substituted drug derivatives. The
alkynyl groups provide reactive sites for further functionalization
of the polymer, for example by reaction with azide derivatives.
Alkynyl and azide groups react via the "click" chemistry mechanism
to form functional groups covalently bonded to the polymer via a
triazole link, for example hydrophilic organic groups, hydrophobic
organic groups, crosslinking groups, and/or drug derivatives. The
polymers can be in the form of unimolecular micelles or crosslinked
nanoparticles. The polymers can further encapsulate a drug for
delivery to a patient (i.e., as compared to drug derivatives that
are covalently attached to the polymer).
As used herein, the term "glycolide" refers to the reaction product
when two mono-basic hydroxy acids form a cyclic diester containing
four (4) carbon atoms and two (2) oxygen atoms in the ring. The
basic ring structure is a dioxandione (i.e., the condensation
product of two glycolic acid (2-hydroxyethanoic acid) molecules).
The glycolide monomer according to the disclosure has an alkynyl
group substituted on at least one (e.g., on one or two) of the
carbon atoms in the ring structure. A poly(glycolide) polymer (or
polyglycolide) includes a polymer resulting from the ring-opening
polymerization of the alkynyl-substituted glycolide monomer. The
alkynyl-substituted glycolide for producing heteropolymers can have
various substituents (e.g., aliphatic (alkyl, alkenyl, etc.),
aromatic (aryl, etc.)) on the carbon atoms along with the alkynyl
groups. A "lactide" is a type of glycolide and refers to the cyclic
diester condensation product of two 3-carbon .alpha.-hydroxy acids,
particularly when the .alpha.-hydroxy acid is lactic acid
(2-hydroxypropanoic acid). A poly(lactide) polymer (or polylactide)
includes a polymer of a lactide.
Poly(Glycolide) Polymers
The poly(glycolide) polymers according to the disclosure can
generally be described in various forms: (1) in terms of repeating
units contained in the polymer, (2) in terms of the polymeric chain
itself, and/or (3) as the reaction product of its precursors.
In a first embodiment, the poly(glycolide) polymer includes one or
more repeating units according to Formula I:
##STR00004## In Formula I: (i) R.sub.1 includes one or more of an
alkynyl group, a hydrophilic organic triazole group, a hydrophobic
organic triazole group, a triazole organic crosslinking group, a
triazole-substituted liquid crystal mesogen, a triazole-substituted
dye, and a triazole-substituted drug derivative; and (ii) R.sub.2
includes one or more of a hydrogen, an alkyl group, an alkynyl
group, a hydrophilic organic triazole group, a hydrophobic organic
triazole group, a triazole organic crosslinking group, a
triazole-substituted liquid crystal mesogen, a triazole-substituted
dye, and a triazole-substituted drug derivative. When x is 0 and
R.sub.2 is unifommly hydrogen, Formula I simply represents an
unmodified poly(glycolide). The polymers according to the
disclosure, however, contain at least some degree of
functionalization; accordingly, x is greater than zero (e.g., x is
between 0 and 1).
In a second embodiment, the poly(glycolide) polymer includes one or
more polymeric chains according to Formula II:
##STR00005## In Formula II: (i) R.sub.1 includes one or more of a
hydrogen, an alkyl group, an alkynyl group, and a triazole organic
crosslinking group; (ii) R.sub.2 includes a hydrophilic organic
triazole group and optionally one or more of a hydrogen, an alkyl
group, an alkynyl group, a hydrophobic organic triazole group, a
triazole-substituted liquid crystal mesogen, a triazole-substituted
dye, and a triazole-substituted drug derivative; and (iii) R
includes a terminal group (e.g., a hydrogen, an alkyl group, for
example a C.sub.1-C.sub.10 alkyl group). In this embodiment, x
ranges from 0 to less than 1, thereby requiring at least some of
the hydrophilic organic triazole group via the R.sub.2 moiety. A
wide variety of degrees of polymerization are acceptable, and n
suitably ranges from about 10 to about 1000 (preferably about 20 to
about 400, or about 20 to about 200).
In a third embodiment, the poly(glycolide) polymer includes a
triazole reaction product of (a) an alkynyl glycolide according to
Formula III
##STR00006## (b) an azide-substituted hydrophilic organic compound;
and, (c) optionally one or more of an azido organic crosslinker, an
azide-substituted hydrophobic organic compound, an
azide-substituted liquid crystal mesogen, an azide-substituted dye,
and an azide-substituted drug. In Formula III: (i) R.sub.1 includes
one or more of a hydrogen, an alkyl group, and an alkynyl group;
(ii) R.sub.2 includes an alkynyl group; and (iii) R includes a
terminal group (e.g., a hydrogen, an alkyl group, for example a
C.sub.1-C.sub.10 alkyl group). Similar to the previous embodiment,
x ranges from 0 to less than 1, and n ranges from about 10 to about
1000 (preferably about 20 to about 400, or about 20 to about
200).
The groups R.sub.1 and R.sub.2 in the forgoing structures can
generally include alkynyl groups. Such groups are generally
hydrocarbons having at least one (e.g., only one) alkynyl
functionality (--C.ident.C--, for example --C.ident.CH) that serves
as a reactive site for the 1,3-dipolar cycloaddition of a
functionalized azide to the polymer backbone, resulting in a
triazole group linking the azide-functional group to the polymer
backbone (described below). The alkynyl groups preferably have 2 to
20 carbons, for example 3 to 12 carbons or 3 to 6 carbons.
Preferably, the alkynyl groups are linear and the alkynyl
functionality is located at the terminal position in the
hydrocarbon chain (i.e., at the furthest position away from the
polymer backbone). For example, a suitable alkynyl group includes
the 1-propynyl (or propargyl) group bonded to the polymer backbone
at the 3-carbon position (e.g., as illustrated in structure 4 of
FIG. 2).
Functionalized Poly(Glycolide) Polymers
The poly(glycolide) polymer can be functionalized to impart desired
features to the basic polymer. The functionalized poly(glycolide)
polymer generally includes the triazole reaction product of the
poly(glycolide) polymer and an azide-substituted organic compound
(at least one of which preferably is a hydrophilic
azide-substituted organic compound). In this case, the groups
R.sub.1 and R.sub.2 in the foregoing structures can additionally
represent the triazole reaction product of the R.sub.1 and R.sub.2
alkynyl groups in the original poly(glycolide) polymer and the
azide-substituted organic compound, for example as illustrated in
structure 6 of FIG. 2 for the case where the azide-substituted
organic compounds are di-azide-substituted alkanes (i.e.,
crosslinkers) and azide-substituted polyoxyethylenes.
The azide-substituted organic compounds are not particularly
limited, generally including hydrophilic organic azido groups,
hydrophobic organic azido groups (also including amphiphilic
organic azido groups), azido organic crosslinking groups (e.g.,
diazido crosslinking groups), azide-substituted drug derivatives,
azide-substituted dye derivatives, and azide-substituted liquid
crystal mesogens, for example including the variety of
representative structures illustrated in FIG. 10. Examples of
suitable azide-substituted organic compounds include
azide-substituted polyoxyalkylenes, azide-substituted organic
amines (and ammonium salts thereof), azide-substituted organic
amides, azide-substituted organic imines, an azide-substituted
carboxylic acid, azide-substituted carbonyl-containing compounds
(e.g., ketones, carboxylic acids, carboxylate salts, esters),
azide-substituted alkyl polyoxyalkylenes, azide-substituted
alcohols, azide-substituted alkanes, azide-substituted alkenes,
diazido alkanes, and azide-substituted dyes. FIG. 10 illustrates
(bottom left structure) a derivative of a rhodamine B dye having an
alkyl azide tether that hydrolyzes in an aqueous environment to
release the dye. FIG. 10 further illustrates (bottom two structures
on right) representative liquid crystal mesogens (i.e.,
azide-substituted molecules exhibiting liquid crystal behavior and
having both a rigid aryl/aromatic portion and a flexible
aliphatic/alkyl portion to which the azido group can be attached).
In some embodiments, the azide-substituted organic compound can
include more than one of the foregoing functional groups, for
example as illustrated in FIG. 10 (e.g., the 3-azidopropyl ester of
butanedioic acid illustrated containing both ester and carboxylic
acids functionalities).
Hydrophilic Organic Triazole Groups: The groups R.sub.1 and R.sub.2
in the forgoing structures can generally include hydrophilic
organic triazole groups. The hydrophilic organic triazole groups
promote the compatibility of the functionalized poly(glycolide)
polymer with water and facilitate the formation of a stable,
unimolecular micelle in an aqueous environment. The hydrophilic
organic triazole groups preferably include reaction products (e.g.,
click reaction products) of a polymer backbone-pendant alkynyl
groups (above) and an azide-substituted polyoxyethylene. For
example, suitable azide-substituted polyoxyethylenes can generally
be polyethylene glycols of the form
N.sub.3[C.sub.2H.sub.40].sub.nR, where n preferably ranges from 2
to 20 (e.g., 3 to 15 or 3 to 8) and R is a terminal group (e.g., a
hydrogen, an alkyl group, for example a C.sub.1-C.sub.10 alkyl
group). Equivalently, the resulting hydrophilic organic triazole
group can be represented by -Tr[C.sub.2H.sub.40].sub.nR, where n
and R are as before, and Tr is a 1,4-disubstituted 1,2,3-triazole
(i.e., linked to the polyoxyethylene at the 1-position of the
triazole and linked to the polymer backbone via the 4-position of
the triazole). A preferred azide-substituted polyoxyethylene is
azidoethyl tetraethylene glycol methyl ether (i.e., n is 5 and R is
methyl in the foregoing generic azide derivative), and the
resulting hydrophilic organic triazole is illustrated in structure
5 of FIG. 2 (top pendant group; for the case when the alkynyl group
is the propargyl group). Other suitable azide-substituted
polyoxyethylenes include PEG-550 monomethyl ether ("mPEG") and
10-axido-2,5,8-trioxadecane ("mDEG", where n is 3 and R is methyl
in the foregoing generic azide derivative). The azide-substituted
polyoxyethylene can be formed by methods generally known in the
art, for example by tosylating a polyoxyethylene (e.g., PEG-550
monomethyl ether or the aforementioned pentaethylene glycol methyl
ether) and reacting the same with an azide salt (e.g., sodium
azide).
As used herein, the term "hydrophilic" as applied to organic
triazole groups can include organic triazole groups (or their
organic azide precursors) that contain substantially exclusively
hydrophilic units. Examples of suitable hydrophilic units include
alkoxy groups (e.g., methoxy, ethoxy (preferable), propoxy, higher
alkoxy), organic amino groups (e.g., primary, secondary, tertiary),
carbonyl groups (e.g., ketones), carboxylic groups (e.g., in acid
and/or salt form, including alkali metal salts), hydroxyl groups,
and esters. The term "hydrophilic" additionally can include
amphiphilic organic triazole groups (and precursors) that contain
both hydrophilic units and hydrophobic units; however, the
amphiphilic organic triazole groups have sufficient hydrophilic
character to form a stable, unimolecular micelle in an aqueous
environment. Thus, the above example azide-substituted
polyoxyethylene of the form N.sub.3[C.sub.2H.sub.4O].sub.nR would
generally be characterized as substantially exclusively hydrophilic
when R is a hydrogen or a methyl group, based on the hydrophilic
nature of the ethoxy --C.sub.2H.sub.4O-- unit; as the number of
carbons in R increases, however, the azide-substituted
polyoxyethylene assumes more hydrophobic character, but would still
be sufficiently hydrophilic (i.e., amphiphilic) to form
unimolecular micelles in an aqueous environment.
The azide-substituted hydrophilic organic groups generally have an
aliphatic hydrocarbon base structure (e.g., linear alkyl) with the
hydrophilic unit(s) located at one or more positions along the
length of the hydrocarbon base structure. The hydrocarbon base
structure can be of variable size depending of the desired
properties of the functionalized polymer; common sizes generally
range from 1 to 50 carbon atoms, for example from 2 to 30 carbon
atoms, 2 to 20 carbon atoms, or 3 to 12 carbon atoms. Examples of
suitable hydrophilic (or amphiphilic) azide-substituted triazole
precursors include azide-substituted polyoxyalkylenes (e.g., 4 to
50 carbon atoms or 6 to 20 carbon atoms, including the
polyoxyethylenes described above), azide-substituted organic amines
and ammonium salts thereof (e.g., 1 to 20 carbon atoms (or 2 to 10
carbon atoms) and having at least one amino/ammonium group),
azide-substituted carboxylic acids/salts thereof (e.g., 1 to 20
carbon atoms (or 2 to 10 carbon atoms) and having at least one
(preferably terminal) carboxylic group), and azide-substituted
alcohols, ketones, ethers, esters, imines, and amides (e.g., 1 to
20 carbon atoms or 2 to 10 carbon atoms). Other, generally
amphiphilic suitable azide-substituted triazole precursors can
include azide-substituted alkyl groups (e.g., 1 to 20 carbon atoms
or 2 to 10 carbon atoms) terminated with polyoxyalkylenes (e.g., 4
to 50 carbon atoms or 6 to 20 carbon atoms). For example, such an
amphiphilic triazole precursor can be of the form
N.sub.3R.sub.a[C.sub.2H.sub.4O].sub.nR, where n and R are as
before, and R.sub.a is an alkyl group (e.g., a C.sub.1-C.sub.20
alkyl group or a C.sub.2-C.sub.10 alkyl group).
Hydrophobic Organic Triazole Groups: The groups R.sub.1 and R.sub.2
in the forgoing structures can include hydrophobic organic triazole
groups in some embodiments (e.g., resulting from the click
functionalization of azide-substituted hydrophobic organic groups).
The hydrophobic organic triazole groups can be included in the
polymer, for example, to adjust the molecular weight of the
polymer, to adjust the hydrophilic/hydrophobic balance in the final
polymer, and/or to obtain lower critical solution temperature
(LCST) behavior. The hydrophobic organic triazole groups also can
be included in the polymer to an extent sufficient to induce the
formation of inverse micelles in a non-aqueous solution (i.e., a
substantially hydrophobic exterior that promotes compatibility with
a non-aqueous solvent and a substantially hydrophilic interior that
promotes, for example, compatibility with an encapsulated
hydrophilic drug or other compound). The azide-substituted
hydrophobic organic groups generally have an aliphatic hydrocarbon,
for example including linear or branched alkyl or alkenyl
hydrocarbons. The hydrophobic aliphatic hydrocarbon can be of
variable size depending on the desired properties of the
functionalized polymer; common sizes generally range from 1 to 50
carbon atoms, for example from 2 to 30 carbon atoms, 2 to 20 carbon
atoms, or 3 to 12 carbon atoms. The hydrophobic organic triazole
groups preferably include reaction products of polymer
backbone-pendant alkynyl groups and azide-substituted alkanes. For
example, suitable azide-substituted alkanes can generally be of the
form N.sub.3[CH.sub.2].sub.nCH.sub.3, where n preferably ranges
from 2 to 20, for example from 4 to 16 or 6 to 14. Equivalently,
the resulting hydrophobic organic triazole group can be represented
by -Tr[CH.sub.2].sub.nCH.sub.3, where n is as before, and Tr is a
1,4-disubstituted 1,2,3-triazole (i.e., linked to the alkane at the
1-position of the triazole and linked to the polymer backbone via
the 4-position of the triazole). Preferred azide-substituted
alkanes are 1-azidobutane, 1-azidodecane, and 1-azidohexadecane
(i.e., n is 3, 9, and 15, respectively; illustrated in FIG. 10).
The hydrophobic organic triazole groups can similarly include
reaction products of polymer backbone-pendant alkynyl groups and
azide-substituted alkenes. The azide-substituted alkenes are
similarly sized to the alkanes, for example including
1-azidooctadec-8-ene (illustrated in FIG. 10). The
azide-substituted alkane/alkene can be formed by methods generally
known in the art, for example by reacting a halogenated
alkane/alkene with an azide salt (e.g., 1-bromodecane with sodium
azide).
Non-Triazole Groups: The groups R.sub.1 and R.sub.2 in the forgoing
structures, when not representing an alkynyl group or a triazole
derivative thereof, can include hydrogen or other organic groups.
The organic group is not particularly limited, generally including
aliphatic hydrocarbon groups (linear or branched, including alkyl
or alkenyl) and/or aromatic (aryl) hydrocarbon groups. The alkyl
groups (and the other non-triazole organic groups in general)
provide a similar functionality to the hydrophobic organic triazole
groups above (e.g., adjust the molecular weight, adjust the
hydrophilic/hydrophobic balance, induce the formation of inverse
micelles). The organic groups differ structurally, however, in that
they are appended directly to the polymer backbone, absent a
triazole linkage. In this embodiment, the organic group is
preferably integrated into the polymer by appending the organic
group to a glycolide monomer. For example, a di-alkyl-substituted
glycolide (e.g., lactide, where the alkyl group is --CH.sub.3) can
be copolymerized with a di-alkynyl-substituted glycolide (e.g.,
structure 3 of FIG. 2). Alternatively or additionally, an
alkyl-substituted derivative of glycolic acid (e.g., lactic acid,
where the alkyl group is --CH.sub.3) can be condensed with an
alkynyl-substituted derivative of glycolic acid (e.g., structure 2
of FIG. 2), thereby forming a blend of a di-alkyl-substituted
glycolide (e.g., lactide), a di-alkynyl-substituted glycolide
(e.g., structure 3 of FIG. 2), and an alkyl-alkynyl-substituted
glycolide (e.g., 3-(2-propynyl)-6-methyl-1,4-dioxane-2,5-dione).
The resulting blend of glycolide derivatives can then be
copolymerized to form a polymer having a desired distribution of
alkyl and alkynyl functionalities. The organic group can have from
0 to 20 carbon atoms (where "0" represents a limiting case in which
the alkyl group is a hydrogen atom), for example 1 to 18 carbon
atoms, 1 to 12 carbon atoms, or 1 to 6 carbon atoms.
Crosslinking Groups: In some embodiments, the poly(glycolide)
polymer can be crosslinked. The crosslinking can be inter- and/or
intramolecular in nature. Crosslinked nanoparticles include
intramolecular crosslinks that link neighboring portions of the
polymer backbone when the polymer is in a micellar configuration,
thereby forming the nanoparticle. Crosslinking is generally
effected by reacting a diazido organic crosslinking compound with
two free alkynyl groups on the polymer backbone, thereby forming a
di-triazole organic crosslinking group between the two polymer
backbone locations. Suitable diazido organic crosslinking compounds
include diazido alkanes, for example
N.sub.3[CH.sub.2].sub.nN.sub.3, where n preferably ranges from 1 to
20, for example from 2 to 10 or 3 to 8. A preferred diazido alkane
is 1,5-diazidopentane, and the resulting crosslinked structure is
illustrated in structure 6 of FIG. 2 (bottom pendant group; for the
case when the alkynyl group is the propargyl group). Other suitable
diazido alkanes include 1,12-diazidododecane (FIG. 10). Other
multi-functional azido organic crosslinking compounds can be used,
for example including triazido functional (or higher) crosslinking
compounds such as triazido (or higher) alkanes (preferably
branched). FIG. 2 (structure 7) and FIG. 4a further qualitatively
illustrate the crosslinking process and structure. Specifically,
structure 7 in FIG. 2 has a crosslinked nanoparticle 10 having
hydrophilic organic triazole groups 12 that outwardly project from
a poly(glycolide) polymer backbone 14 forming the interior of the
crosslinked nanoparticle 10. The crosslinks 16 represent the
di-triazole organic crosslinking groups between different regions
of the polymer backbone 14. The diazido alkanes can be formed, for
example, reacting a di-halogenated alkane with an azide salt (e.g.,
1,5-dibromopentane with sodium azide).
Drug Delivery: The poly(glycolide) polymer can be used as a vehicle
for the controlled release of a drug. In some embodiments, the any
of a variety of biologically relevant drugs can be covalently
attached to the polymer, for example when the polymer is in the
form of a polymeric chain, a unimolecular micelle, or a crosslinked
nanoparticle. A particular drug of interest is functionalized using
conventional methods so that the drug molecule is azide-substituted
(e.g., halogenation or tosylation of an alkyl group followed by
reaction with an azide salt, synthesis of aromatic azides from the
corresponding amines by reaction with t-butyl nitrite and
azidotrimethylsilane, ring-opening of epoxides to form 1,2-azido
alcohols, etc.). The drug is then attached to the polymer as the
triazole reaction product of the azide-substituted drug and a free
pendant alkynyl group along the polymer backbone. In general, any
drug that can be modified to include a pendant azide without
destroying (or otherwise substantially inhibiting) the activity of
the drug can be used. Preferably, an azide tether (i.e., an azide
group with an optional linking group between the azide group and
the drug) is used that is able to hydrolyze to reveal the drug. For
example, an azide tether can include an alkyl (or other
hydrocarbon) group having an ester linkage to the drug at one end
of the alkyl group and an azide group at the opposing end of the
alkyl group, thus allowing ester hydrolysis to release the drug in
an aqueous environment (e.g., as illustrated by the drug surrogate
rhodamine B azide derivative in FIG. 10).
Poly(glycolide) Copoloymers: In some embodiments, the
poly(glycolide) polymer can be a copolymer, for example a copolymer
incorporating other biodegradable repeating units. The inclusion of
a comonomer can be used to alter the density of functional groups
(i.e., alkynyl groups and/or triazole derivatives thereof) along
the polymer backbone while retaining the biodegradable character of
the polymer as a whole. A preferred copolymer includes a
poly(glycolide-lactide) copolymer that further includes lactide
repeating units with glycolide repeating units along the polymer
backbone (e.g., as described above for embodiments in which R.sub.1
and/or R.sub.2 include alkyl groups). Other suitable copolymer
repeating units include polyester units having from 2 to 10 carbon
atoms in the repeating unit along the length of the polymer
backbone (e.g., including the repeating unit derived from
copolymerization with .epsilon.-caprolactone). In such a case, the
copolymer can be a block copolymer (e.g., when the poly(glycolide)
polymer is further reacted with a lactide monomer) or a random
copolymer (e.g., when a glycolide monomer such as propargyl
glycolide is reacted with a lactide or other monomer).
Polymer Characteristics: In the foregoing embodiments, the
poly(glycolide) polymer generally includes a variety of groups
along its backbone, for example hydrogens, alkynyl groups,
hydrophilic organic triazole groups, hydrophobic organic triazole
groups (also including amphiphilic organic triazole groups),
di-triazole organic crosslinking groups, and triazole-substituted
drug derivatives. The various groups can be included in the polymer
in various proportions according to desired properties of the final
polymer. The polymer backbone requires at least some alkynyl groups
to facilitate subsequent polymer functionalization via triazole
formation. Thus, while some of the backbone groups can include
non-functional hydrogens, the alkynyl glycolide precursor (e.g., as
represented by Formula III) preferably is saturated with alkynyl
groups (i.e., all or substantially all of the R.sub.1 and R.sub.2
in Formula III are alkynyl groups).
In a preferred embodiment, the poly(glycolide) polymer includes a
substantial fraction of hydrophilic organic triazole groups at its
functional positions R.sub.1 and R.sub.2. For example, preferably
about 50% to about 95% of the functional positions R.sub.1 and
R.sub.2 available in the polymer are occupied by one or more types
of hydrophilic organic triazole groups, more preferably about 60%
to about 90% (or about 65% to about 85%), for example about 75%.
Within the context of Formula I, for example, this corresponds to a
structure in which (i) R.sub.1 represents hydrophilic organic
triazole groups and x ranges from about 0.5 to about 0.95 (or about
0.6 to about 0.9, about 0.65 to about 0.85, or about 0.75), and
(ii) R.sub.2 represents non-hydrophilic organic triazole groups
(e.g., unreacted alkynyl groups, di-triazole organic crosslinking
groups, triazole-substituted drug derivatives). In a further
embodiment, the remaining functional positions R.sub.1 and R.sub.2
(i.e., those that include alkynyl groups not converted to
hydrophilic organic triazole groups) can be crosslinked and/or used
as sites for covalent drug attachment. For example, about 10% to
about 100% of the available alkynyl groups remaining after
functionalization with the hydrophilic organic triazole groups can
be crosslinked with di-triazole organic crosslinking groups.
Alternatively, about 5% to about 50% of the functional positions
R.sub.1 and R.sub.2 available in the polymer are occupied by
di-triazole organic crosslinking groups, more preferably about 10%
to about 40%, or about 15% to about 35%. When the final
poly(glycolide) polymer is to include a triazole-substituted drug
derivative, the polymer is not saturated with the diazido organic
crosslinker to preserve some alkynyl groups for drug
attachment.
In another embodiment, the poly(glycolide) polymer includes a lower
fraction of hydrophilic organic triazole groups at its functional
positions R.sub.1 and R.sub.2 to permit a higher degree of
incorporation of non-hydrophilic functional groups (e.g.,
hydrophobic groups (whether attached directly to the polymer
backbone or via a triazole linkage), liquid crystal mesogens
attached via a triazole linkage). For example, about 10% to about
50% of the functional positions R.sub.1 and R.sub.2 available in
the polymer can be occupied by hydrophilic organic triazole groups,
for example about 15% to about 45% (or about 20% to about 40%).
Similarly, about 10% to about 50% of the functional positions
R.sub.1 and R.sub.2 available in the polymer can be occupied by
non-hydrophilic functional groups, for example about 15% to about
45% (or about 20% to about 40%). In any event, a portion of the
functional positions R.sub.1 and R.sub.2 still can be utilized for
crosslinking and/or drug attachment, for example at levels similar
to the foregoing embodiment containing a substantial fraction of
hydrophilic organic triazole groups.
Unimolecular Polymer Configurations
The poly(glycolide) polymer can assume a unimolecular micellar
configuration in an aqueous solution when the polymer includes
pendant hydrophilic organic triazole groups. The unimolecular
micelle self-aggregates and configurationally rearranges in the
aqueous solution such that the micelle has a substantially
hydrophilic exterior (e.g., resulting from the outwardly protruding
hydrophilic organic triazole groups on the polymer backbone) and a
substantially hydrophobic interior (e.g., resulting from the
polymer backbone occupying the interior of the micelle). The
unimolecular micelle can be suitably formed by slowly adding a
solution of the poly(glycolide) polymer in a water-soluble organic
solvent (e.g., acetone) to cold water (e.g., less than about
10.degree. C.) with stirring, followed by in vacuo removal of the
solvent. Absent the crosslinks 16, structure 7 of FIG. 2 and
structure 1 of FIG. 5a illustrate the unimolecular micelle.
The poly(glycolide) polymer can be further formed into a
crosslinked nanoparticle by reacting residual alkynyl groups in the
unimolecular micelle with di-triazole organic crosslinking groups.
The resulting structure is similar to that of the unimolecular
micelle: a unimolecular polymer particle having a substantially
hydrophilic exterior and a substantially hydrophobic interior,
along with the addition of intramolecular crosslinks in the
particle interior (i.e., as illustrated in structure 7 of FIG. 2).
Crosslinking has the effect of locking the resulting nanoparticle
into a micellar configuration with a hydrophobic interior and a
hydrophilic exterior. The degree of crosslinking further affects
the rate of release of a drug from the poly(glycolide) polymer to
the surrounding environment. More specifically, an increased degree
of crosslinking generally reduces the rate of drug release.
However, when the degree of crosslinking is greater than about 10%
(relative to total functionalization sites in the polymer), the
drug hydrolysis rate of triazole-bound drugs is likely to be
relatively slow and not further limited by the degree of
crosslinking. Thus, a controlled-release composition including the
poly(glycolide) polymer and the drug can be designed to release a
drug at a desired rate by controlling the degree of crosslinking.
In a further refinement, the poly(glycolide) polymer-drug
combination can be provided as a plurality of crosslinked
nanoparticles with a distribution of crosslinking degrees selected
to control the rate of release of an encapsulated component or a
covalently bound component in the plurality of crosslinked
nanoparticles. For example, a fraction of the plurality can be
relatively lightly crosslinked to promote rapid drug release, while
another fraction can be relatively highly crosslinked to promote
long-term drug release. Suitable degrees of crosslinking (relative
to total functionalization sites in the polymer) can range from
greater than 0% (i.e., lightly crosslinked) to about 20%, 30%, or
40% (i.e., highly crosslinked), for example about 1% to about 20%,
30%, or 40%, or about 2% to about 15%. For example, when the degree
of crosslinking is about 20% and a di-functional organic
crosslinker is used, an amount of organic crosslinker equal to
about 10% (on a number/molar basis) of the total alkynyl
functionalization sites in the original polymer is reacted with the
polymer (i.e., thus taking into account that most organic
crosslinker molecules will generally be able to form two triazole
bonds based on their dual azide functionality). Additionally,
different degrees of crosslinking can be associated with different
drugs such that a time-dependent drug delivery profile also can be
drug-dependent (e.g., a first drug is released from the plurality
at a first time after administering the composition and a second
(different) drug is released at a second (different) time after
administering the composition).
The unimolecular micelles and/or the crosslinked nanoparticles
generally have a diameter of about 100 nm or less. The small size
is useful in drug-delivery applications, for example facilitating
target cell entry and/or biodegradation. For example, the
unimolecular micelles generally have a diameter of about 50 nm or
less (or about 15 nm to about 50 nm) as measured by dynamic light
scattering in aqueous solution. The corresponding crosslinked
nanoparticles are generally slightly smaller, having a diameter of
about 35 nm or less (or about 8 nm to about 35 nm), also as
measured by dynamic light scattering in aqueous solution.
Alternatively, the micellar/nanoparticle diameters can be
characterized by the TEM measurement of the corresponding
dehydrated particles. In this case, the unimolecular micelles and
the crosslinked nanoparticles generally have a diameter of about 15
nm or less (or about 2 nm to about 10 nm, for example about 4 nm to
about 8 nm) as measured by TEM.
For drug-delivery applications, drugs can be combined with the
polymer in a variety of ways. A drug generally includes any
chemical administered to a living being (e.g., human patient or
other animal) to treat, cure, prevent, and/or diagnose a disease or
other physical/mental condition of the living being. As described
above, an azide-substituted drug derivative can be covalently bound
to the polymer in any of its various forms, for example: (i) a
polymer in solution in an organic solvent (e.g., acetone), (ii) a
polymer in a unimolecular micellar configuration in an aqueous
solution, or (iii) a polymer in a crosslinked nanoparticle
configuration. In any of these cases, the drug is covalently bound
to the polymer backbone via a triazole link. Additionally, an
unmodified (i.e., non-azide substituted) drug can be encapsulated
in the hydrophobic interior of a unimolecular micelle. For example,
the organic-solvent solution of the poly(glycolide) polymer can
additionally contain a drug (e.g., a substantially hydrophobic
drug); as the organic solvent is removed from an aqueous solution
containing the polymer and the drug, the drug becomes encapsulated
within the hydrophobic interior of the forming unimolecular
micelle. Once formed, the unimolecular micelle can be further
crosslinked while retaining the encapsulated drug within the
interior of the resulting crosslinked nanoparticle.
Thus, the foregoing poly(glycolide) polymers can generally be used
in a method to deliver a drug compound to a cell (e.g., human
(preferable), other animal, plant). The method generally includes
(a) providing the drug compound with the poly(glycolide) polymer,
and (b) releasing the drug compound to the cell over a period of
time. The method can be applied either in vivo or in vitro, and the
poly(glycolide) polymer can be either in the form of a unimolecular
micelle, a crosslinked nanoparticle, or both (e.g., as an
additional manner of controlling drug release rate). The drug
compound can be encapsulated by the poly(glycolide) polymer, in the
form of a triazole-substituted drug derivative covalently bound to
the poly(glycolide) polymer, or both (e.g., when multiple drugs are
to be delivered by polymer composition). In an embodiment, the drug
compound is in dosage unit form with a carrier.
EXAMPLES
The following Examples illustrate the disclosed compositions and
methods, but are not intended to limit the scope of any claims
thereto.
In the Examples, the following techniques were used. Polymer
molecular weights were determined by GPC-MALS (Multi-Angle Light
Scattering) at 35.degree. C. using two PLgel 10.mu. mixed-B columns
in series, and THF as the eluting solvent at a flow rate of 1
mL/min. An Optilab rEX (Wyatt Technology Co.) was used as a
detector, calculating absolute molecular weights using a DAWN EOS
18 angle light scattering detector (Wyatt Technology Co.) with a
laser wavelength of 684 nm. All samples were filtered through a 0.2
.mu.m Whatman PTFE syringe filter prior to GPC analysis. .sup.1H
NMR analyses were carried out at room temperature on a Varian
UnityPlus-500 spectrometer at 500 MHz or a Varian Unity Plus-300 at
300 MHz referenced to the solvent residual proton chemical shifts.
.sup.13C NMR spectra were obtained on Varian UnityPlus-500 at 125
MHz or a Varian UnityPlus-300 at 75 MHz. UV-vis spectra were
recorded with a Cary 300 Bio WinUV, Varian spectrophotometer.
Example 1
Synthesis of Poly(Alkynyl Glycolide) Precursor Polymer
General procedure for bulk polymerizations. Solvent-free
polymerizations were carried out in sealed ampoules prepared from
3/8 in. diameter glass tubing that were charged with the monomers
and a stir bar, and connected to a vacuum line through a vacuum
adapter. After evacuating the ampoule for 4 hours, the ampoule was
filled with nitrogen and a syringe was used to add a predetermined
amount of tin(II) 2-ethylhexanoate (Sn(Oct).sub.2) and
4-t-butylbenzyl (BBA) alcohol solutions to the ampoule through the
adapter. The solvent was removed in vacuo and the ampoule was
flame-sealed and immersed in oil bath at 125.degree. C. At the end
of the polymerization the ampoule was cooled, opened, and the
polymer was dissolved in dichloromethane. A portion of the sample
was evacuated to dryness and analyzed by NMR for conversion. The
rest of the polymer solution was precipitated from cold
methanol.
Representative polymerization of propargyl glycolide. With
reference to the structures and synthesis route illustrated in FIG.
2, 2-Hydroxy-4-pentynoic acid ethyl ester (structure 1),
2-hydroxy-4-pentynoic acid (structure 2),
3,6-di-2-propynyl-1,4-dioxane-2,5-dione (structure 3), and
poly(propargyl glycolide) (structure 4; "poly(PGL)") were prepared
as described in Jiang et al., Macromolecules, 41, 1937-1944 (2008),
which is incorporated herein by reference in its entirety.
Propargyl glycolide (0.65 g) was polymerized for 45 min of a
[M]/[I]=150. The conversion of monomer to polymer calculated from
.sup.1H NMR was 97%. Precipitation and drying under vacuum gave
0.64 g poly(propargyl glycolide) as a light brown solid (98%).
.sup.1H NMR (CDCl.sub.3) .delta. 5.31-5.46 (br, 1H), 2.79-3.03 (br
m, 2H), 2.01-2.18 (br, 1H). GPC-MALS (THF): Mn=31,000 g/mol,
PDI=1.28.
Example 2
Synthesis of Unimolecular Micelles
To move from amphiphilic comb polymers to cross-linked
nanoparticles, azidoethyl tetraethylene glycol methyl ether was
appended to a fraction (e.g., about 75%) of the available alkynyl
groups in the poly(PGL) base polymer using the below "click"
reaction conditions to give poly(PGL.sub.n[EG.sub.yX.sub.100-y]),
where n represents the average degree of polymerization, y
represents the fraction of pendant alkynes reacted with the
azide-functionalized polyoxyethylene, and 100-y represents the
remaining fraction of unreacted pendant alkynes. For example,
poly(PGL.sub.124[EG.sub.75X.sub.25]) describes a poly(PGL) chain of
degree of polymerization 124, and having 75% of its pendant alkynes
reacted with the azidoethyl tetraethylene glycol methyl ether
(resulting structure 5 in FIG. 2). The azidoethyl tetraethylene
glycol methyl ether was formed by tosylating pentaethylene glycol
methyl ether and then reacting the product with sodium azide.
General procedure for "click" functionalization. The desired amount
of acetylene functionalized polymer, 0.5-0.8 eq. of the
azide-substituted polyoxyethylene (relative to the number of
available alkynyl groups in the base polymer), and sodium ascorbate
powder (12 mol. % with respect to the alkynyl groups) were
dissolved in 5 mL DMF in a Schlenk flask, and the solution was
deoxygenated by three freeze-pump-thaw cycles. After the solution
warmed to room temperature a 0.1 M solution of CuSO.sub.4.5H.sub.2O
in deoxygenated DMF (5 mol % with respect to the acetylene groups)
was added under nitrogen; the reaction mixture was stirred at room
temperature for 3 hours. At the end of the reaction, the solids in
the reaction mixture were removed by filtration and ion exchange
resin beads (AMBERLITE IRC-748 ion-exchange resin) were added to
the solution for 6 hours to remove residual copper. The beads were
removed by filtration, the DMF removed in vacuo and the polymer was
isolated by dialysis (MWCO=12-14,000) first in an acetone/water
(1:1) solution, and then by dialysis in Milli-Q water, and then
dried under vacuum.
Poly(PGL.sub.124-[EG.sub.75X.sub.25]). Poly(PGL) (50 mg) (M.sub.n,
GPC-MALS=23,800 g/mol, PDI=1.10) and azidoethyl tetraethylene
glycol methyl ether (0.11 g; 0.75 eq with respect to the alkynyl
groups) were dissolved in 5 mL DMF for the click reaction.
Poly(PGL.sub.124[EG.sub.75X.sub.25]) was isolated as a viscous
liquid (58 mg, 70%).
Formation of unimolecular micelles. A solution of
poly(PGL.sub.124[EG.sub.75X.sub.25]) (20 mg) in 1 mL acetone was
slowly added drop-wise to stirred ice-cold Milli-Q water (20 mL) in
a Schlenk flask. The solution was allowed to stir for 30 minutes
before the acetone was removed in vacuo, thereby forming
unimolecular micelles in the remaining water.
Micelle characterization. Micelles were characterized using
GPC-MALS, .sup.1H NMR, AFM, TEM and Dynamic light scattering (DLS).
The results for representative samples are reported below. DLS data
were obtained using a temperature-controlled Protein Solutions Dyna
Pro-MS/X system. All samples were filtered through a 0.2 .mu.m
Whatman PTFE syringe filter and allowed to equilibrate in the
instrument for 15 minutes at 25.degree. C. before measurements were
taken. The uniformity of the particle sizes were determined by a
monomodal curve fit, which assumes a single particle size with a
Gaussian distribution.
Surface profile measurements were performed with a Pacific
Nanotechnology Nano-R atomic force microscope in close contact
(oscillating) mode to generate height images that were not altered
other than a simple leveling procedure. Silicon tips with a spring
constant of 36 N/m, tip curvature of 10-20 nm, and a resonance
frequency of 286-339 kHz were used for all experiments. The
nanoparticles were dispersed in Milli-Q water and the solutions
(typical concentration, 20-300 .mu.g/ml) were spin coated at 5000
rpm for 40 sec on the silicon substrate.
Transmission electron microscopy samples (typical concentration,
20-300 .mu.g/mL) were dropped onto nickel grids pretreated with
FORMVAR resin and allowed to settle for 2 min before removing the
excess solution. The sample was negatively stained with either a 1%
phospho-tungstic acid (PTA) stain or 2% uranyl acetate (UA) stain
prepared with deionized water. Micrographs were collected at
270,000-370,000.times. magnification on a JEOL 100CX TEM.
Various poly(glycolide) polymers according to the disclosure were
formed by reacting poly(PGL) with azidoethyl tetraethylene glycol
methyl ether to form polymers of variable molecular weight and
fractional conversion of alkynyl groups to triazole-linked
hydrophilic organic groups. The polymers were then formed in into
unimolecular micelles. The characteristics of the various resulting
unimolecular micelles are summarized in Table 1. Additionally,
FIGS. 3a and 3b are AFM and TEM images, respectively, of a sample
of unimolecular micelles (polymer 2a from Table 1 below).
TABLE-US-00001 TABLE 1 Micelle Characterization Data M.sub.n
M.sub.n R.sub.h Height.sub.avg Polymer poly(PGL).sup.a PDI.sup.a
N.sub.alkyne.sup.b poly(PGL.sub.n[EG.sub- .NX.sub.1-N]).sup.a
(nm).sup.c (nm).sup.d 1a 10,000 (n .apprxeq. 52) 1.19 74 31,300 35
.+-. 2 3.2 .+-. 0.1 2a 23,800 (n .apprxeq. 124) 1.30 85 69,000 35
.+-. 2 3.0 .+-. 1 3a 31,000 (n .apprxeq. 161) 1.28 66 90,000 37
.+-. 2 3.0 .+-. 0.1 .sup.adetermined by GPC-MALS using THF as the
eluting solvent at 35.degree. C.; parenthetical value for n of
poly(PGL) represents approximate degree of polymerization using a
MW = 192 for the propargyl glycolide repeating unit. .sup.bpercent
of alkynyl groups reacted with azide-substituted polyoxyethylene,
determined by NMR spectroscopy. .sup.chydrodynamic radius,
determined by dynamic light scattering at 25.degree. C.
.sup.ddetermined by AFM; 0.20 mg/mL solution spin coated in Si
wafer.
Example 3
Synthesis of Crosslinked Nanoparticles
Cross-linking was effected by dissolving the polymer in a minimal
amount of acetone followed by drop-wise addition of the solution to
stirred Milli-Q water. The acetone was removed in vacuo, and then
cross-linked nanoparticles were formed by coupling
1,5-diazidopentane to the residual alkynes of the poly(PGL) base
polymer, as schematically illustrated in FIG. 4a. The progress of
the crosslinking reaction was monitored by following the
disappearance of the alkyne stretching band in the IR spectrum
(FIG. 4c). NMR analysis of the particles following their
purification by dialysis, first in an acetone/water solution and
then by dialysis in Milli-Q water, showed no evidence for residual
azides or alkynyl groups. Thus, substantially all of the original
alkynyl groups in the poly(alkynyl glycolide) precursor polymer
(i.e., poly(PGL) in this case) were consumed/functionalized in the
final crosslinked nanoparticles. FIG. 4b shows a TEM image of the
cross-linked nanoparticles; further AFM/TEM images are shown in
FIGS. 4d and 4e. Given the polymer molecular weight of 72,000 g/mol
("polymer 2c") and assuming a spherical shape and a density of 1.0
g/cm.sup.3 for a dried particle, a fully dense particle was
calculated to have a 6 nm diameter, consistent with the 6.+-.2 nm
diameter-seen in TEM. While the agreement may be somewhat
fortuitous, their uniformity is consistent with particles derived
from individual polymer chains.
Core cross-linked nanoparticles. A round bottom flask equipped with
a magnetic stir bar and charged with
poly(PGL.sub.124[EG.sub.75X.sub.25]) micelles (20 mL of 0.20 mg/mL
solution) and sodium ascorbate (1 eq with respect to the remaining
acetylene groups) and CuSO.sub.4.5H.sub.2O (0.5 eq with respect to
the remaining alkynyl groups), and then placed under nitrogen. The
solution was allowed to stir 5 min and then a solution of
1,5-diazidopentane (10-100 mol. % with respect to the remaining
alkynyl groups) dissolved in 1 mL acetone was slowly added
drop-wise. The solution was allowed to stir under nitrogen 2 d, and
was then condensed and dissolved in 5 mL of DMF. Amberlite IRC-748
ion-exchange resin beads were added, and, after 6 h, the beads were
removed, and the solution was transferred to presoaked dialysis
tubing (MWCO ca. 12-14 kDa). The polymer was purified by dialysis
first in an acetone/water (1:4) solution and then by dialysis in
Milli-Q water.
As illustrated in FIG. 4c, the disappearance of the alkyne C--H
stretch (3340-3270 nm) and the appearance of the triazole stretch
(N--H) (3300-2500 nm) were observed using infared spectroscopy. IR
spectroscopy also confirmed that no additional azide groups (2100
nm) were present. The spectra of poly(PGL) and
poly(PGL.sub.124[EGyX.sub.100-y]), with varying degrees of
functionalization were taken with Mattson Galaxy 3000 FT-IR.
Various crosslinked nanoparticles according to the disclosure were
formed by reacting the unimolecular micelles with
1,5-diazidopentane to form polymers of variable molecular weight
and degrees of crosslinking. The polymers were then formed in into
unimolecular micelles. The characteristics of the various resulting
crosslinked nanoparticles are summarized in Table 2. In Table 2,
polymers 1b, 2b, and 3b are the crosslinked analogs to polymers 1a,
2a, and 3a in Table 1, respectively, and the Mn values are listed
prior to crosslinking.
TABLE-US-00002 TABLE 2 Nanoparticle Characterization Data Mn.sup.a
% cross- Polymer poly(PGL.sub.n[EG.sub.yX.sub.100-y]) linked.sup.b
R.sub.h (nm).sup.c H.sub.avg (nm).sup.d 1b 31,300 50 18 .+-. 2 4.5
.+-. 0.3 2b 69,000 100 12 .+-. 2 6.0 .+-. 1 3b 90,000 10 39 .+-. 2
3.5 .+-. 0.8 .sup.adetermined by GPC-MALS using THF as the eluting
solvent at 35.degree. C. .sup.bpercent of remaining alkynes that
were cross-linked by 1,5-diazidopentane; determined by initial feed
ratio. .sup.chydrodynamic radius, determined by dynamic light
scattering at 25.degree. C. .sup.ddetermined by AFM; 0.20 mg/mL
solution spin coated in Si wafer.
Estimation of cross-linked nanoparticle size. One polymer,
M.sub.n=72,000 g/mol (from GPC).times.1 mol/6.02.times.10.sup.23
chains=1.19.times.10.sup.-19 g. Assume the polymer can be
approximated as a sphere of density (.rho.)=1 g/cm.sup.3 Mass of a
dense particle=V.times..rho.=(4/3).pi.r.sup.3.times.1
g/cm.sup.3=1.13.times.10.sup.-19 g. Diameter of a
1.13.times.10.sup.-19 gparticle with .rho.=1
g/cm.sup.3=2.times.radius=((3/4)V/.pi.).sup.1/3 1
nanoparticle.apprxeq.6 nm.
Example 4
Covalent Attachment of Small Molecules to Micelles and
Nanoparticles
A question with any cross-linking protocol is the success and
degree of crosslinking in the particles. To answer this question
and to explore the possibility of loading crosslinked unimolecular
micelles with covalently attached biologically relevant drugs, an
azido-substituted drug surrogate was covalently attached to the
synthesized polymer. Specifically, 3-azido-7-hydroxycoumarin, a dye
precursor that transforms to a fluorescent dye after undergoing a
1,3-dipolar cycloaddition reaction.sup.27, was used as the
surrogate. As illustrated in FIG. 5a, two click reactions paths
using poly(PGL.sub.124[EG.sub.75X.sub.25]) as the substrate and the
dye precursor were considered. The first reaction (path "a" in FIG.
5a) evaluated the potential for covalent attachment of small
molecules, and the second reaction (path "b" in FIG. 5a) probed the
extent of crosslinking using an excess of the dye precursor to
react with residual alkynyl groups in the nanoparticles. This is
important for drug delivery, because residual alkynyl groups
represent potential site of drug attachment. The relative dye
loadings were interrogated by measuring the fluorescence
intensities of 1.times.10.sup.-6 M nanoparticle solutions. The
results indicate that the nanoparticles can be loaded with a
covalently bonded triazole-substituted drug derivative after being
formed into crosslinked nanoparticles, and the efficiency of the
click functionalization was determine based on the number residual
alkynyl groups after functionalization.
Covalent attachment of 3-azido-7-hydroxycoumarin to nanoparticles.
Using poly(PGL.sub.124[EG.sub.75X.sub.25]) as the substrate and the
conditions described earlier, two reactions were performed: (1) a
single click step using an equimolar mixture of the dye precursor
and 1,5-diazidopentane (path "a" in FIG. 5a); and (2) a two-step
process where 5%, 10%, or 100% of the remaining alkynyl groups of
poly(PGL.sub.124[EG.sub.75X.sub.25]) were first click-crosslinked
with 1,5-diazidopentane, followed by a second click reaction using
an excess of the dye precursor (path "b" in FIG. 5a). The
nanoparticle fluorescence was recorded using a Fluorolog-3
(Instruments S.A., Inc.) fluorometer. All samples were diluted to
1.times.10.sup.-6 M using Milli-Q water, excited at
.lamda..sub.ex=440 nm, and the fluorescence emission was observed
from 450-500 nm. FIG. 5b illustrates the fluorescence intensity of
the following 1-step and 2-step click reactions on
poly(PGL.sub.124[EG.sub.75X.sub.25]) substrates: a single click
step using an equimolar mixture of the dye precursor and
1,5-diazidopentane (blue; line 1); a two step process where 5% of
the remaining alkynyl groups were crosslinked, followed by a second
click reaction using an excess of the dye precursor (red; line 2);
the same protocol with 10% crosslinking (orange; line 3); and the
same protocol with 100% crosslinking (green; line 4).
Example 5
Drug Encapsulation by Unimolecular Micelles
Controlled drug release improves drug efficacy, reduces toxicity
and improves patient compliance and convenience..sup.28 A similar
protocol was used to evaluate encapsulation of hydrophobic
molecules in nanoparticles. Poly(PGL.sub.124[EG.sub.75X.sub.25])
and azobenzene, a drug surrogate, were dissolved in a minimal
amount of acetone, and dispersed in Milli-Q water. After removal of
the acetone in vacuo, unimolecular micelles were formed, and the
encapsulation of azobenzene in the micelles was confirmed by
azobenzene's characteristic absorbance at 320 nm, as illustrated in
FIG. 6. Specifically, FIG. 6 illustrates the UV-vis spectra of:
azobenzene-loaded unimolecular polymeric micelles (blue; line 1),
unimolecular polymeric micelles (green; line 2), and azobenzene in
water (red; line 3).
Example 6
Drug Encapsulation by Crosslinked Nanoparticles
The drug encapsulation experiment of Example 5 was repeated, but
this time using click chemistry to crosslink the azobenzene-loaded
unimolecular polymeric micelles with 1,5-diazidopentane, thereby
forming azobenzene-loaded crosslinked nanoparticles. To track the
release of azobenzene from the particles, crosslinked nanoparticle
solutions were placed in dialysis tubing (MWCO=12-14 kDa) and
dialyzed with Milli-Q water. Removing aliquots of the nanoparticle
solution from dialysis bag and measuring their UV-vis spectra
allowed monitoring of the continuous release of encapsulated
azobenzene over 10 hours. As shown in FIG. 7, the azobenzene
release rate from the cross-linked nanoparticles was about the rate
measured for a non-crosslinked control (i.e., non-crosslinked
unimolecular polymeric micelles). These data are consistent with
successful crosslinking, and suggest that an ensemble of particles
having different degrees of crosslinking can be the basis of a
drug-release composition that maintains long-term controlled
release strategy.
Example 7
Biocompatibility of Crosslinked Nanoparticles
For in vivo applications, nanoparticles must be biocompatible and
degradable. In a single click reaction,
poly(PGL.sub.124[EG.sub.75X.sub.25]) was crosslinked and a
rhodamine dye was appended to the nanoparticles. After dialysis to
remove residual dye and click reagents, cortical astrocytes were
cultured in a 10 .mu.g/mL solution of dye-labeled nanoparticles.
After 24 hours, the cells were washed to remove particles that had
not entered the cells and then imaged by confocal microscopy. FIG.
8a illustrates cortical astrocytes cultured in media containing 10
.mu.g/ml of crosslinked nanoparticles for 24 hours. In FIG. 8a, the
red color emanates from rhodamine dye covalently linked to the
nanoparticles that have entered cells. FIG. 8a shows that the
nanoparticles readily entered the cells, probably due to their
small size and their polyoxyethylene exterior, which screens
particle/protein interactions. The cells remained healthy for more
than 30 days and showed no signs of nanoparticle toxicity.
These data are in agreement with the viability assays on various
crosslinked nanoparticles, which show no detectable toxicity for
nanoparticle concentrations up to about 3000 .mu.g/mL, as
illustrated in FIG. 8b. Specifically, HeLa cells were plated in
96-well black-clear bottom plates at 10,000 cells/well in 100 .mu.L
of culture medium lacking antibiotic and grown for 22 hr at
37.degree. C. Cells were about 70%-80% confluent based on
microscopy. Nanoparticle solutions were prepared in OptiMEM
(Invitrogen) when dilutions were required (50 .mu.l total volume)
and added to the 100 .mu.L of culture medium. Cells were incubated
for an additional 22 hr and then 20 .mu.L Cell Titer Blue (CTB)
reagent was added. Fluorescence was quantified in a fluorescence
plate reader 2 hr after CTB reagent was added, according to the
manufacturer's instructions. Results are reported as the
average.+-.standard deviation (n=3). Non-crosslinked nanoparticles
(poly(PGL.sub.124[EG.sub.75X.sub.25]; unimolecular micelles or
"NonCL NP" as referenced in FIG. 8b) and crosslinked nanoparticles
(poly(PGL.sub.124[EG.sub.75X.sub.25] with 10% cross-linking of
remaining alkynyl groups; "CL NP") correspond to polymers mentioned
in the text. LipoFectamine ("LF2k"), linear poly(ethylene imine)
(M.sub.n=25,000; "LPEI"), and poly(L-lysine) (M.sub.n=150,000;
"PLL") were used as controls. LF2k is reported as dilutions of the
purchased solution. FIG. 8b compares the cell toxicity (in
normalized relative fluorescence units) for the foregoing
nanoparticles at varying concentrations.
Example 8
Hydrolytic Degradation of Functionalized Poly(glycolide)
Poly(glycolide) and related polymers are susceptible to hydrolytic
degradation. Degradation experiments were performed at 36.degree.
C. in Milli-Q water, and the measured molecular weights were
plotted in FIG. 9 (open squares:
poly(PGL.sub.124[EG.sub.75X.sub.25]); open triangles:
rac-poly(lactide). The molecular weights according to the random
chain scission model (i.e., (M.sub.n(0)/M.sub.n(t)-1)/P.sub.n(0)
versus time, where P.sub.n(0) is the initial degree of
polymerization) were also plotted in FIG. 9 (dashed lines). The
polyoxyethylene-functionalized poly(propargyl glycolide) degrades
more rapidly than rac-lactide, as shown in FIG. 9. Accordingly,
combinations of the two polymers (e.g., as random or block
copolymers) can be used to control the rate of degradation.
Degradation of poly(PGL.sub.124[EG.sub.75X.sub.25]). 10 mg samples
of non-crosslinked poly(PGL.sub.124[EG.sub.75X.sub.25]) were placed
in 20 mL culture tubes, and 10 mL of Milli-Q water was added. The
tubes were capped and placed in a degradation chamber at 36.degree.
C. Samples were removed at predetermined times, dried, and the
molecular weight was determined by GPC-MALS. For comparison, a
similar experiment was run looking at the degradation of
rac-lactide; however, the experiment was run at 55.degree. C. to
accelerate degradation. The results were normalized to account for
the initial degree of polymerization (P.sub.n(0)) of each
polymer.
Example 9
Synthesized Crosslinked Nanoparticle Compositions
The various azide-substituted functional groups generally described
above (and those specifically illustrated in FIG. 10) can be
incorporated into a poly(glycolide) polymer. Several crosslinked
nanoparticle compositions have been synthesized according to the
disclosure to illustrate the variety of functional groups (and the
variety in the levels of incorporation of the functional groups)
that can be added to a poly(glycolide) polymer backbone having
alkynyl functionality via the click chemistry mechanism. Table 3
presents the composition of the synthesized nanoparticle
compositions.
TABLE-US-00003 TABLE 3 Crosslinked Nanoparticle Compositions ID
Composition V.18
PG.sub.131[(C.sub.1EO.sub.4C.sub.2).sub.80CL.sub.20] V.32
PG.sub.65[PEG.sub.63Rh.sub.4CL.sub.33] V.92
PG.sub.177[PEG.sub.48CL.sub.25] V.93
PG.sub.177[PEG.sub.30A1.sub.20CL.sub.25] V.121
PG.sub.300[PEG.sub.47C10.sub.32CL.sub.20] VII.22
PG.sub.157[PEG.sub.80CL.sub.10] VII.108
PG.sub.300[PEG.sub.37.5LC.sub.37.5CL.sub.24] VII.126
PG.sub.300[PEG.sub.30LC.sub.30CL.sub.25]
For the compositions in Table 3, the polymer (PG) was first
synthesized according to the general procedure outlined in the
above examples. The ligands (PEG, CL, A1, C10, LC,
C.sub.1EO.sub.4C.sub.2, and Rh) were then added to polymer chain,
also as described above. The structures of the specific polymer and
ligands are illustrated in FIG. 11. The formulae in Table 3
describe the composition of the particles. For example,
nanoparticle V.93 is a poly(propargyl glycolide) with 177 repeat
units, and PEG.sub.30Al.sub.20CL.sub.25 denotes that 30% of the
pendent alkynes of the polymer were reacted with the PEG-based
azide, 20% with the amine A1, and 25% of the pendent alkynes of the
polymer were reacted with the crosslinker CL. Some polymers were
partially modified, meaning that some alkynyl groups remained
unreacted in the nanoparticle (e.g., VII.22 has 80% PEG, 10% CL,
and the remaining 10% of the alkynyl groups in the original PG
polymer remain unreacted in the nanoparticle). The Rh groups in the
nanoparticles act as markers. Specifically, the Rh dye is released
from the nanoparticle in an aqueous environment (i.e., allowing the
time-release properties of the nanoparticles to be monitored, for
example in relation to a triazole-linked drug to be delivered by a
nanoparticle). Liquid crystal mesogens can be added to the
nanoparticles to act as a visible marker that remains attached to
the nanoparticle in an aqueous environment (i.e., allowing the
mobility properties of the nanoparticles to be monitored).
Because other modifications and changes varied to fit particular
operating requirements and environments will be apparent to those
skilled in the art, the disclosure is not considered limited to the
example chosen for purposes of illustration, and covers all changes
and modifications which do not constitute departures from the true
spirit and scope of this disclosure.
Accordingly, the foregoing description is given for clearness of
understanding only, and no unnecessary limitations should be
understood therefrom, as modifications within the scope of the
disclosure may be apparent to those having ordinary skill in the
art.
Throughout the specification, where the compositions, processes, or
apparatus are described as including components, steps, or
materials, it is contemplated that the compositions, processes, or
apparatus can also comprise, consist essentially of, or consist of,
any combination of the recited components or materials, unless
described otherwise. Combinations of components are contemplated to
include homogeneous and/or heterogeneous mixtures, as would be
understood by a person of ordinary skill in the art in view of the
foregoing disclosure.
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